Small ubiquitin-like modifier (SUMO) conjugation modulates all major cellular pathways, including those associated with gene expression and genome stability, protein quality control, proteasomal degradation of proteins and DNA damage repair. Transient cerebral ischemia massively activates SUMO conjugation, resulting in a dramatic rise in levels of SUMO2/3-conjugated proteins. Cell culture studies suggest that the post- ischemic activation of SUMO2/3 conjugation is a protective stress response. However, the role of SUMO conjugation in the fate of post-ischemic neurons in the intact brain and the mechanisms and pathways that link SUMO conjugation to restoration of function impaired by transient ischemia are not known. Without this knowledge it is highly unlikely that the SUMO conjugation pathway can be manipulated for therapeutic purposes. Our long-term goal is to understand how to manipulate the SUMO conjugation pathway for preventive and therapeutic purposes. The objective of this particular application is to elucidate the role of individual SUMO paralogues in the recovery of neurons from ischemic stress and to identify the mechanisms and pathways involved. The central hypothesis is that SUMO conjugation plays a key role in modulating path- ways that are critical for death/survival decisions in post-ischemic neurons. This hypothesis has been formulated on the basis of data produced in our laboratory. The rationale for the proposed studies is that after we have verified the protective role of SUMO conjugation in post-ischemic neurons in vivo and have identified the underlying mechanisms and pathways, we will have established an important platform for designing new strategies for preventive and therapeutic interventions in clinically relevant pathological states associated with a transient episode of insufficient blood supply. Based on strong preliminary data and the development of novel SUMO transgenic and knockout animals in our laboratory, the hypothesis will be tested by pursuing the following specific aims: 1) Characterize new SUMO transgenic and knockout mouse models; 2) Determine the effects of individual SUMO paralogues on post-ischemic neuronal cell damage and functional recovery; 3) Determine how SUMO conjugation is linked to the fate of post-ischemic neurons; 4) Determine how transient ischemia affects the crosstalk between ubiquitin and SUMO conjugation. The approach is innovative because it is the first study to use SUMO transgenic and knockout animals and to per- form proteomic analyses to determine the role of SUMO conjugation in cerebral ischemia. The proposed research is significant, because we expect to uncover the mechanisms that link SUMO conjugation to the viability and function of post-ischemic neurons. Ultimately, such knowledge is expected to translate into new strategies for therapeutic intervention in pathological states associated with an episode of insufficient blood supply and in other disorders associated with the SUMO conjugation pathway, including diabetes, heart failure, and degenerative diseases.